Difference between revisions of "Part:BBa K4665170:Design"

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===Design Notes===
 
===Design Notes===
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After extracting the sequence from the plasmid, T7 exonuclease digestion will need to be performed in order to get ssDNA needed for DNA origami.
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The transformation of the dsDNA scaffold sequence into the desired ssDNA scaffold for folding was accomplished by following the procedure outlined by Noteborn et al. (2021). To achieve this, the dsDNA scaffold was amplified using a standard primer in combination with a modified primer containing 5 phosphorothioate linkages, thus introducing these linkages at the 5' end of the desired scaffold strand. Subsequent to PCR, the phosphorothioate-modified scaffold can be selectively digested with T7 exonuclease. This enzyme will degrade the strand lacking phosphorothioate linkages from the 5' to the 3' end, while the phosphorothiate-modified strand remains protected against T7 exonuclease digestion.
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===Source===
 
===Source===

Revision as of 16:28, 8 October 2023


Octahedron DNA-origami (3024bp long; 42bp edges)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 919
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 919
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 738
    Illegal BglII site found at 1310
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 919
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 919
    Illegal NgoMIV site found at 132
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The transformation of the dsDNA scaffold sequence into the desired ssDNA scaffold for folding was accomplished by following the procedure outlined by Noteborn et al. (2021). To achieve this, the dsDNA scaffold was amplified using a standard primer in combination with a modified primer containing 5 phosphorothioate linkages, thus introducing these linkages at the 5' end of the desired scaffold strand. Subsequent to PCR, the phosphorothioate-modified scaffold can be selectively digested with T7 exonuclease. This enzyme will degrade the strand lacking phosphorothioate linkages from the 5' to the 3' end, while the phosphorothiate-modified strand remains protected against T7 exonuclease digestion.

Source

The plasmid with the 3024bp sequence was obtained from addgene. The original plasmid was constructed by Nafisi et al., (2018). They converted a pUC18 vector into a pagemid for custom ssDNA production by adding four components: 1) a full-length M13 origin for ssDNA initiation. 2) Kpnl and BamHl restriction sites for insert cloning. 3) M13 PS for phage particle export. 4) modified M13 origin to serve as the ssDNA synthesis terminator.

References

Nafisi, P. M., Aksel, T., Douglas, S. M. (2018) Construction of a novel phagemid to produce custom DNA origami scaffolds, Synthetic Biology, Volume 3, Issue 1, https://doi.org/10.1093/synbio/ysy015